Lymphocytes are mediators of normal tissue inflammation as well as pathologic tissue damage such as occurs in rheumatoid arthritis and other autoimmune diseases. In order to fully exploit the antigenic repertoire of the immune system, vertebrates have evolved a mechanism for distributing lymphocytes with diverse antigenic specificities to spatially distinct regions of the organism [Butcher, E. C., Curr. Top. Micro. Immunol. 128, 85 (1986); Gallatin et al., Cell 44, 673 (1986); Woodruff et al., Immunol. Today 10, 23 (1989); Yednock et al., Adv. Immunol. 44, 313 (1989)].
This mechanism involves the continuous recirculation of the lymphocytes between the blood, where the cells have the greatest degree of mobility, and the lymphoid organs, where the lymphocytes encounter sequestered and processed antigen.
It has been recognized for some time that the trafficking of lymphocytes from the blood into secondary lymphoid organs, such as lymph nodes (LN) and gut-associated Peyer's patches (PP), is initiated by an adhesive interaction with the specialized endothelial cells of high endothelial venules (HEV) [Berg et al., Immunol. Rev. 108, 5 (1989); Duijvestijn and Hamann, Immunol. Today 10. 23 (1989); Woodruff et al., Annu. Rev. Immunol. 5, 201 (1987); Yednock and Rosen, Adv. Immunol. 44, 313 (1989); Stoolman, Cell 56, 907 (1989)]. Considerable evidence indicates that the lymphoid organ-selective migration or "homing" of lymphocytes is dictated in large part by organ-specific binding of lymphocytes to HEV [Butcher (1986), Supra]. Operationally, the lymphocyte-associated molecules underlying the organ-selective interaction with HEV are termed "homing receptors" while the cognate endothelial molecules are known as "HEV ligands" [Gallatin et al. (1986), Supra; Rosen, Curr. Opin. Cell. Biol. 1, 913 (1989)]. The endothelial HEV ligands are postulated to be distinctive for the different lymphoid organs and as such are proposed to be responsible for regulating the lymphocyte populations to enter each class of lymphoid organ [Butcher, Am. J. Pathol. 136, 3 (1990)]. A characterization of the detailed molecular mechanisms underlying lymphocyte trafficking is interesting from both a scientific and a clinical standpoint, since similar adhesive processes may be involved in both the normal and pathogenic forms of leukocyte inflammation [Watson et al., Nature 349, 164-167 (1991)].
Of the homing receptors, the most thoroughly studied is a receptor initially termed peripheral lymph node homing receptor (pnHR). This receptor was first defined in the murine system by the MEL-14 monoclonal antibody (mAb), an antibody that was found to recognize an about 90 kD leukocyte surface antigen (referred to as gp90.sup.MEL) [Gallatin et al., Nature 303, 30 (1983)]. This antibody was found to block the lymphocyte adhesion to HEV of peripheral and mesenteric lymph nodes in the Stamper-Woodruff in vitro adherence assay and to prevent in vivo migration to lymph nodes. A homing function for gp90.sup.MEL was definitely shown by the finding that detergent solubilized and soluble recombinant forms of the receptor can selectively block adhesive sites for lymphocytes on LN but not those on PP HEV [Geoffroy and Rosen, J. Cell. Biol 109, 2463 (1989)].
Molecular cloning of cDNAs encoding the murine and human gp90.sup.MEL receptors revealed a transmembrane protein with a calcium-type (C-type) lectin domain at its extracellular amino terminus, followed by an EGF motif, two complement regulatory motifs related to those found in proteins with complement-binding activity, a transmembrane domain, and a short cytosolic tail [Lasky et al., Cell 56. 1045 (1989); Siegelman et al., Science (Wash., D.C.) 243, 1165 (1989); Siegelman et al., Proc. Natl. Acad. Sci. USA 86, 5562 (1989); Tedder et al., J. Exp. Med. 170 (1) 123 (1989); Tedder et al., J. Immunol. 144. 532 (1989), Bowen et al., J. Cell Biol. 109, 421 (1989); Camerini et al., Nature 342(6245), 78 (1989); copending application Ser. No. 315,015 filed 23 February 1989; WO 91/08298 published 13 Jun. 1991].
Other researchers identified another molecule associated with neutrophil adhesion. It was proposed that this molecule, termed the endothelial leukocyte adhesion molecule ELAM-1, is an inducible adhesion molecule whose role may be to mediate the attachment of neutrophils to venular endothelial cells adjacent to sites of inflammation [Bevilacqua et al., Proc. Natl. Acad. Sci. USA 84, 9238 (1989); Hession et al., Proc. Natl. Acad. Sci. USA 87(5), 1673 (1990)].
Investigations of the proteins contained in the alpha granules of platelets led to the discovery of a further adhesion molecule variously termed granular membrane protein-140 (GMP-140), platelet activation dependent granule external membrane protein (PADGEM) or CD62 [McEver et al., J. Biol. Chem. 259, 9799 (1984); Bonfanti et al., Blood 73, 1109 (1989); Hattori et al., J. Biol. Chem. 264(14), 7768 (1989)]The cDNA sequence encoding this receptor was determined by Johnston et al., Cell 56, 1033 (1989).
Comparison of their amino acid sequences revealed that these three adhesion molecules are related in a highly striking and compelling manner. Their common mosaic structure consists of a calcium dependent lectin or carbohydrate-binding motif, an epidermal growth factor-like (EGF) motif, and variable numbers of a complement regulatory (CR) motif. The ordered conjunction of these motifs has given rise to the name LEC-CAM (Lectin Egf Complement regulatory-Cell Adhesion Molecule) for this new family of leukocyte endothelial cell adhesion molecules [Stoolman, Cell 56:907 (1989)]Alternatively, the name "selectin" has been applied to this family [Bevilacqua et al., Science 243:1160 (1989); Geng et al., Nature 343:757 (1990)].
The three members of the LEC-CAM or selectin family of cell adhesion molecules are: L-selectin (a.k.a. peripheral lymph node homing receptor (pnHR), LEC-CAM-1, LAM-1, gp90.sup.MEL, gp100.sup.MEL, gp110.sup.MEL, MEL-14 antigen, Leu-8 antigen, TQ-1 antigen, DREG antigen), E-selectin (LEC-CAM-2, LECAM-2, ELAM-1) and P-selectin (LEC-CAM-3, LECAM-3, GMP-140, PADGEM). These receptors will selectin family members and of the genes encoding them are illustrated in FIGS. 1 and 2, respectively.
The finding that simple monomeric sugars, such as mannose-6-phosphate (M6P) and fructose-1-phosphate, can block the interactions of murine and human lymphocytes with HEV of peripheral lymph nodes (pln) [Stoolman et al., J. Cell Biol. 96. 722 (1983); Stoolman et al., J. Cell Biol. 99, 1535 (1984); Stoolman et al., Blood 70, 1842 (1987)] suggested that the endothelial ligand recognized by L-selectin is carbohydrate-based. In one series of experiments, Rosen and colleagues demonstrated that the homing receptor-dependent binding of lymphocytes to pln HEV was abolished by either in vitro or in vivo treatment with broad spectrum sialidases [Rosen et al., Science (Wash., D.C.) 228. 1005 (1985); Rosen et al., J. Immunol. 142, 1895 (1989)]. Because this enzyme selectively removes terminal sialic acid residues from oligosaccharides, these results strongly implied that sialic acid was a critical element for recognition.
The nature of the endothelial molecule(s) recognized by L-selectin was subsequently probed with a unique recombinant chimera, consisting of the extracellular domain of L-selectin joined to the hinge, CH2 and CH3 regions of the human IgGI heavy chain [see WO 91/08298 published 13 Jun. 1991 for the chimera, and Watson et al., J. Cell Biol. 110, 2221 (1990) for its use as a probe for adhesive ligands of lymph node high endothelial venules]. Initial studies with this so-called receptor-immunoglobulin chimera demonstrated that it could adhere to (a) peripheral and mesenteric lymph node-specific HEV ligand(s) in cell blocking and immunohistochemical experiments [Watson et al. (1990), Supra]. The immunohistochemical recognition of this HEV ligand was abolished by treatment of lymph node sections with sialidase, suggesting that a component of the carbohydrate recognized by L-selectin was sialic acid-like and further accentuated the importance of the lectin domain in L-selectin-mediated adhesion [Rosen et al., Science (Wash. D.C.) 228, 1005-1007 (1985); Rosen et al. (1989), Supra, and True et al., J. Cell Biol. 11, 2757-2764 (1990)]. These results demonstrated the specificity of the L-selectin-immunoglobulin chimera for the pln HEV ligand and established that the ligand expresses carbohydrate residues that are essential for homing receptor-mediated cell adhesion.
A recent series of publications confirmed that the E-selectin ligand also has a carbohydrate character. Several laboratories, adopting a wide range of approaches, have concluded that an E-selectin ligand is a carbohydrate known as sialyl Lewis.sup.x (sLex) or a closely related structure known as CD65 or VlM-2 [NeuAca2-3Galbl-4(Fucal-3)GlcNAcbl]. Lowe et al.[Cell 63, 475 (1990)], transfected non-myeloid cells With an a1,3/4 fucosyltransferase and generated ligand activity for E-selectin, which was correlated with the expression of the Slex determinant. Goeltz et al. [Cell 63(6), 1349 (1990)]identified and cloned an a1,3 fucosyltransferase that appeared to be involved in the synthesis of the actual ELAM-1 ligand in myeloid cells. Using more direct approaches, Phillips et al. [Science 250(4984), 1130 (1990)] and Walz et al. [Science 250(4984), 1132 (1990)]were able to show inhibition of E-selectin dependent adhesion with either Slex-containing glycoconjugates or antibodies to Slex. The critical participation of both the sialic acid and fucose moieties in ligand activity were demonstrated in these studies. Finally, Tiemeyer et al. [Proc. Natl. Acad. Sci. USA (1991)]purified several myeloid-derived glycolipids that had ligand activity for E-selectin transfected cells in a solid-phase assay. Mass spectroscopic analysis of purified, E-selectin binding glycolipid revealed that the minimal structure necessary for activity was a silylated lactosamine with a second internal N-acetyllactosamine unit containing an a1,3-linked fucose on the N-acetylglucosamine (CD65). As was true for the Slex determinant, both the sialic acid and fucose were essential for binding activity of the putative ligand.
Progress has also been made in the identification of ligands for P-selectin. Larsen et al. [Cell 63, 467 (1990)]have implicated the Lex determinant [Galbl-4(al-3Fuc)GlcNAc] as an important element of the P-selectin ligand on myeloid cells. However, sialic acid is also required for full ligand activity, probably in an a2,3 linkage [Corral et al., Biochem. Biophys. Res. Commun. 172. 1349 (1990); Moore et al., J. Cell Biol. 112, 491 (1991)]. There is a possibility that the ligand for P-selectin is the same or very similar to that for E-selectin, especially since both selectins bind to a very similar spectrum of cells types [Polley et al., Proc. Natl. Acad. Sci. USA 88, 6224 (1991)].
The remarkable homology in selectin structures as well as the already demonstrated similarities in the ligands suggest that the ligands will have related and yet subtlely different structures.
An object of the present invention is to provide a method for the purification of a selectin ligand.
Another object of the invention is to provide purified selectin, specifically L-selectin ligands.
A further object of the present invention is to provide nucleic acid sequences encoding selectin glycoprotein ligands.
It is another object to determine the amino acid sequences of the selectin ligands, and to identify the (0- and N-linked) glycosylation sites on these ligands.
A still further object is to enable the preparation of amino acid sequence and/or glycosylation variants of selectin ligands, not otherwise found in nature.
In a still further aspect, the invention provides a method of designing selectin inhibitors, mimicking carbohydrate based determinants of the selectin ligands.
These and further objects of the present invention will be apparent for one skilled in the art.